Ex vivo method for determining potential GLP-2 receptor modulators

ABSTRACT

Disclosed herein is a method for measuring the contractility of intestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Also disclosed is an assay which directly measures the activity of GLP-2 or GLP-2 ligands ex vivo and permits the screening of putative GLP-2 ligands in native tissue.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 13/154,071, filed on Jun. 6, 2011, which issued as U.S. Pat.No. 8,257,983 on Sep. 4, 2012, which is a divisional application of U.S.patent application Ser. No. 12/358,123 filed on Jan. 22, 2009, whichissued as U.S. Pat. No. 7,977,061 on Jul. 12, 2011, which is adivisional application of U.S. patent application Ser. No. 11/607,030,filed on Dec. 1, 2006, which issued as U.S. Pat. No. 7,498,141 on Mar.3, 2009 which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/741,075 filed on Dec. 1, 2005, nowexpired, the entire contents of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Glucagon-like peptide-2 (GLP-2) is a 33-amino acid proglucagon-derivedpeptide secreted by the endocrine L-cell primarily in the lowergastrointestinal tract in response to luminal nutrients. Plasma levelshave been shown to significantly increase within an hour of ingesting ameal, in particular following the ingestion of carbohydrates or fat (1).GLP-2 has been shown to be responsible for the regulation ofproliferation and apoptosis of the intestinal epithelium (2,3). Thesechanges in part result in an increase in mucosal surface area, enhancedabsorptive efficiency and barrier function in the small intestine (4-6).GLP-2 also decreases gastric motility, inhibits gastric acid secretion,increases nutrient transport activity and acutely increases intestinaland portal blood flow (7-11).

GLP-2 and GLP-2 analogues promote the growth and repair of theintestinal epithelium in models of disease, including enhancedadaptation and nutrient absorption following small bowel resection andalleviation of TPN-induced hypoplasia in rodents (12-15). GLP-2analogues have demonstrated decreased mortality and improvement ofdisease-related histopathology in animal models of intestinal damagesuch as indomethacin-induced enteritis, dextran sulfate-induced colitisand chemotherapy-induced mucositis (16-19).

The intestinotrophic effects of GLP-2 are mediated by the GLP-2 receptor(GLP-2R), a member of the superfamily of G-protein coupled receptors andmost closely related to the GLP-1 and glucagon receptor gene subfamily(20). The GLP-2R is a high-affinity, ligand-specific functional receptorcoupled to the G-protein Gs. Studies of the activation of the clonedGLP-2R by GLP-2 analogues show a correlation of in vitro activity within vivo intestinotrophic efficacy (20).

To date, characterization of GLP-2R function has been limited toheterologous cell line expression, mucosal fractions and primary cellcultures. GLP-2 selectively stimulates cAMP production in recombinantlyexpressing GLP-2R cell lines, isolated intestinal mucosal fractionscontaining enteroendocrine and neural cells and primary hippocampalcultures (21-24). The study of native GLP-2R biological activity hasfocused on in vivo animal models and ex vivo model systems that requireexogenous application of GLP-2 to the whole animal prior to tissueisolation (2-19, 25-31). There is no ex vivo method for characterizingputative GLP-2 ligands directly; such a method would have obviousutility, for example in the screening and characterization of GLP-2ligands of pharmaceutical interest.

SUMMARY OF THE INVENTION

Disclosed herein is a method for measuring the contractility ofintestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Thus, oneaspect of the invention is an assay which directly measures the activityof GLP-2 or GLP-2 ligands. Another aspect of the invention is the directcharacterization of the GLP-2 receptor. A further aspect of theinvention is a method for screening compounds for activity at the GLP-2receptor, such as putative GLP-2 agonists, antagonists, modulators orthe like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the organ bath trace of the effect of 1 uM GLP-2 (1-33) onrat contractility in duodenum, jejunum, ileum and colon.

FIG. 2 shows the organ bath trace of the effect of GLP-2 (1-33) on ratcolon contractility.

FIG. 3 Shows the organ bath trace of the effect of [gly2]GLP-2 on ratcolon contractility.

FIG. 4 shows the inhibitory effects of GLP-2 analogues on spontaneouscontractility in rat colon.

FIG. 5 shows the effect of GLP-2 analogues on cAMP accumulation inEBNA293 cells stably expressing rat GLP-2R.

FIG. 6 shows the effect of GLP-2 (3-33) on GLP-2 (1-33)-inducedinhibition of colon contractility.

FIG. 7 shows the effect of GLP-2 (3-33) on teduglutide-inducedinhibition of rat colon contractility.

FIG. 8 shows the effect of a small molecule antagonist on GLP-2(1-33)-induced inhibition of colon contractility.

FIG. 9 shows the effect of a small molecule positive modulator on GLP-2(1-33)-induced cAMP accumulation.

FIG. 10 shows the effect of a small molecule positive modulator on GLP-2(1-33)-induced inhibition of colon contractility.

DETAILED DESCRIPTION OF THE INVENTION Definitions

hGLP-2R is the human GLP-2 receptor; rGLP-2R is the rat GLP-2 receptor.hGLP-2 is human GLP-2 (also known as Teduglutide); rGLP-2 is rat GLP-2.

A “GLP-2 ligand” is defined as any molecule which interacts with theGLP-2 receptor; such molecules may be peptides (such as analogues ofnaturally occurring GLP-2) or small molecules.

A “modulator” is defined as a molecule which modulates the activity of aGLP-2 agonist.

“Intestinal tissue” is broadly defined as intestine from the pylorus tothe rectum. “Small intestine” is defined as intestine from the pylorusto the ileo-cecal valve (it may also be defined as duodenum, jejunum andileum). “Large intestine” is defined as intestine distal from the cecum(it may also be defined as colon tissue).

Disclosed herein is an assay which, for the first time, demonstrates theability of GLP-2 receptor ligands to directly affect nativeGLP-2R-mediated effects in colon tissue ex vivo. As a result, it ispossible to directly screen and pharmacologically characterize GLP-2ligands, such as peptidic analogies of GLP-2, as well as small moleculeagonists, antagonists and modulators, against whole tissue.

Such tissue is of mammalian origin; in one aspect it is of rat origin,in others it is of mouse or guinea-pig origin.

The assay method comprises measuring the contractility of segments ofintestinal tissue ex vivo. The fact that this assay is useful for thestated purpose is not obvious, based upon the known expression levels ofthe GLP-2 receptor in this tissue. Further, previous studies have shownthat direct application of GLP-2 ex vivo to this tissue has no effect.

One aspect of the invention is a method for characterizing the GLP-2receptor; for example in an endeavour to better understand thephysiological role of GLP-2 and the GLP-2 receptor in intestinal tissueor to elucidate the role of putative mediators related to GLP-2activation and physiology.

Another aspect of the invention is a method for determining whether acompound is active on the GLP-2 receptor comprising the step ofmeasuring contractility of intestinal tissue segments ex vivo in thepresence and absence of compound, wherein a difference in contractilityin the presence of the compound is indicative of activity on the GLP-2receptor.

Yet another aspect of the invention is a method for determining whethera compound acts as a GLP-2 receptor agonist comprising the step ofmeasuring contractility of intestinal tissue segments ex vivo in thepresence and absence of compound, wherein inhibition of contractility inthe presence of the compound is indicative of GLP-2 receptor agonistactivity.

Yet another aspect of the invention is a method for determining whethera compound acts as a GLP-2 receptor antagonist comprising the step ofmeasuring the effect of a GLP-2 receptor agonist on contractility ofintestinal tissue segments ex vivo in the presence and absence of thecompound, wherein a decrease of agonist inhibition of contractility inthe presence of the compound is indicative of GLP-2 receptor antagonistactivity.

Another aspect of the invention is a method of assaying compounds foractivity at the GLP-2 receptor comprising the steps of:

i. Obtaining a segment of intestinal tissue,

ii. Suspending the segment in an organ bath,

iii. Placing the segment under tension,

iv. Incubating the segment with a compound of interest and

v. Measuring the contractility of the tissue; wherein an effect oncontractility of the segment is indicative of activity on the GLP-2receptor.

In one aspect the tissue is intestinal tissue. In another it is smallintestine tissue. In yet another it is large intestine tissue or colontissue. FIG. 1 shows the Organ Bath Trace of the effect of 1 uM GLP-2(1-33) on rat contractility in duodenum, jejunum, ileum and colon. FIG.2 shows that GLP-2 (1-33) inhibits spontaneous colon contractility in aconcentration-dependent manner in rat colon tissue; FIG. 3 shows theanalogous effect of Teduglutide.

The assay of putative agonists may be conducted by incubation of thecolon segments with a predetermined concentration of the ligand to betested for 5 to 10 minutes. FIG. 4 shows typical results for a number ofGLP-2 analogues. FIG. 5 shows the effect of GLP-2 analogues on cAMPaccumulation in EBNA293 cells stably expressing rGLP-2R.

The assay of putative antagonists may be conducted by pre-incubation ofthe colon segments with a predetermined concentration for, for example,5 to 10 minutes of the ligand to be tested followed by addition andincubation of a predetermined concentration of a GLP-2 agonist. FIGS. 6,7 and 8 show typical results for a number of GLP-2 antagonists;concentration-response curves are clearly right-shifted, as expected.

The assay of putative modulators, may be conducted by pre-incubation ofthe colon segments with a predetermined concentration of the ligand tobe tested for, for example, 5 to 10 minutes followed by the addition andincubation of a pre-determined concentration of a GLP-2 agonist. FIG. 10show typical results for a GLP-2 positive modulator; the effect of themodulation is clearly demonstrated. FIG. 9 shows the correspondingeffect on GLP-2 (1-33)-induced cAMP accumulation.

Methods & Results

It should be noted that the methods described below are illustrative innature, and that the invention is not limited to the particularembodiments described.

Segments of colon from male Sprague Dawley rats were suspended in organbath chambers containing Krebs' solution and maintained at 37° C. with95% O₂ and 5% CO₂. Basal tone, contractility rate and peak height wereused to measure the properties of spontaneous and ligand-mediatedcolonic contractions. Segments were initially loaded to a tension of 2 gand allowed to equilibrate for 60 minutes. During this period, segmentswere repeatedly washed every 10 minutes. [gly2]GLP-2 (“Teduglutide”, aGLP-2 agonist) and GLP-2 analogues were subsequently incubated with eachtissue segment for 5 to 10 minutes. The tissues were allowed tore-equilibrate with repeated washes following each experiment.

Teduglutide and GLP-2 (1-33) inhibited muscle contractility in aconcentration-dependent manner with an estimated IC₅₀ of 4.9±1.9 (SE) nM(n=6) and 13.5±5.4 nM (n=9), respectively. While having no effect alone,GLP-2 (3-33) (a GLP-2 antagonist/partial agonist) blocked the inhibitoryeffects of Teduglutide and GLP-2. In the presence of 10 μM GLP-2 (3-33),Teduglutide and GLP-2 (1-33) inhibited colon contractility with an IC₅₀of 59.9±11.9 nM (n=6) and 142.7±55.9 nM (n=5) respectively. The rankorders of potency of GLP-2 peptide analogues were similar to thatpreviously reported for their cAMP activation in cell linesrecombinantly expressing the rGLP-2R.

These studies demonstrate the ability of GLP-2 to reproducibly andsignificantly reduce spontaneous contractility of rat colon segmentssuspended in organ bath chambers. GLP-2-induced inhibition of coloncontractility is concentration-dependent and reversible following awash-out period. The GLP-2R antagonist/partial agonist GLP-2 (3-33)blocks the effect of GLP-2 (1-33) in a concentration-dependent manner.The rank order of potency of GLP-2 analogues on the inhibition of coloncontractility was comparable to the rank order of increased cAMPaccumulation in heterologous cells expressing GLP-2R.

Therefore the described assay enables native GLP-2R characterization bythe direct application of GLP-2 to intestinal tissue segments ex vivo.As a result, it is possible to screen and pharmacologically characterizeGLP-2 peptide analogues, GLP-2R small molecule agonist and antagonistsagainst whole tissue.

Colon Contractility—Methods

Animals

Male Sprague-Dawley (SD) rats (Charles River Canada, Quebec), weighing200-250 g, were used for these studies. The animals were housed oncontact bedding and kept with free access to food (Purina Rodent Diet)and water in a facility with regulated temperature and humidity, underan alternate 12-hour light/dark cycle. Animals were acclimatized for 3˜5days prior to harvesting of tissues.

Tissue Preparation

Rats were euthanized by decapitation. The entire intestine was quicklyremoved and placed in a Petri dish containing a Sylgard layer and 37° C.Krebs' solution bubbled with 95% O₂ and 5% CO₂. The Krebs' solution (pH7.4) contained 118.1 mM NaCl, 25 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄,1.2 mM MgSO₄.7H₂O, 2.5 mM CaCl₂.2H₂O and 11 mM glucose. The colon wasisolated, cleaned of superficial fat and mesentery and sectioned intoeight 1-cm long segments. The lumen was cleaned by carefully flushingeach segment with Krebs' solution using plastic transfer pipets.Segments were transferred to a separate Petri dish containing freshKrebs' solution and suture threads were tied to both ends of eachsegment. Tissues were suspended longitudinally using surgical suture,one end attached to the transducer on the top and the other to thetissue holder at the bottom of 20 ml organ bath chambers containingKrebs' solution, maintained at 37° C. with 95% O₂ and 5% CO₂. Isometrictensions were continuously measured using calibrated force displacementtransducers and data was collected and processed by the MP100WSW systemwith AcqKnowledge software

Experiment Protocol

Segments of colon were initially loaded to a tension of 2 g and allowedto equilibrate for a 60 min period. During this period, segments werewashed with Krebs' solution at 10 minutes intervals. When the tissuesobtained regular and stable contraction patterns, GLP-2 agonist responseexperiments were conducted by the addition of eight concentrations ofcompound into eight separate organ baths for 5-10 minutes. Routinely,one-hour washes were interspersed between repeat applications ofcompounds to allow for recovery. The effect of GLP-2 (3-33) on GLP-2agonist analogues were conducted by pre-incubating colon segments with 1or 10 μM GLP-2 (3-33) for 5-10 minutes. Then agonist responses weremeasured at various concentrations as described above.

Data Analysis

Tension was measured by changes in peak height. Data were calculated andexpressed as percentage response relative to the basal level (prior tothe experiment). Graphs and IC₅₀ determinations were plotted by thepercentage response in GraphPad Prism (v 3.02). In order to assess thesignificance of the effect of GLP-2 and Teduglutide paired t-tests wereconducted on the Log [IC₅₀] values.

REFERENCES

-   1. Xiao Q., Boushey R. P., Drucker D. J., Brubaker P. L. Secretion    of the intestinotrophic hormone glucagon-like peptide 2 is    differentially regulated by nutrients in humans. Gastroenterol.    1999; 117:99-105-   2, Drucker D. J., Ehrlich P., Asa S. L., Brubaker P. L. Induction of    intestinal epithelial proliferation by glucagon-like peptide-2.    Proc. Natl. Acad. Sci. USA 1996; 93:7911-16-   3. Tsai C.-H., Hill M., Drucker D. J. Biological determinants of    intestinotrophic properties of GLP-2 in viva. Am. J. Physiol. 1997;    272:828-32-   4. Tsai C. H., Hill M., Asa S. L., Brubaker P. L., Drucker D. J.    Intestinal growth-promoting properties of glucagon-like peptide-2 in    mice. Am. J. Physiol. 1997; 273:E77-84-   5. Brubaker P. L., Izzo A., Hill M., Drucker D. J. Intestinal    function in mice with small bowel growth induced by glucagon-like    peptide-2. Am. J. Physiol. 1997; 272:E1050-8-   6. Benjamin M. A., McKay D. M., Yang P.-C., Cameron H., Perdue, M H.    Glucagon-like peptide-2 enhances intestinal epithelial barrier    function of both transcellular and paracellular pathways in the    mouse. Gut 2000; 47:112-9-   7. Wojdemann M., Wettergren A., Hartmann B., Hoist J. J.    Glucagon-like peptide-2 inhibits centrally induced antral motility    in pigs. Scand. J. Gastroenterol. 1998; 33:828-32-   8. Wojdemann M., Wettergren A., Hartmann B., Hilsted L., Hoist J. J.    Inhibition of sham feeding-stimulated human gastric acid secretion    by glucagon-like peptide-2. J. Clin. Endocrinol. Metab. 1999;    84:2513-17-   9. Guan X., Stoll B., Lu X., Tappenden K. A., Hoist J. J., Hartmann    B., Burrin D. GLP-2-Mediated Up-Regulation of Intestinal Blood Flow    and Glucose Uptake Is Nitric Oxide-Dependent in TPN-Fed Piglets.    Gastroenterol. 2003; 125: 136-147-   10. Cheeseman C. I., Tsang R. The effect of gastric inhibitory    polypeptide and glucagon like peptides on intestinal hexose    transport. Am. J. Physiol. 1996; 271:G477-G482-   11. Cheeseman, C. I. Upregulation of SGLT-1 transport activity in    rat jejunum induced by GLP-2 infusion in vivo. Am. J. Physiol. 1997;    273:R1965-R1971-   12. Scott R. B., Kirk D., MacNaughton W. K., Medding J. B. GLP-2    augments the adaptive response to massive intestinal resection in    rat. Am. J. Physiol. 1998; 38:G911-21-   13. Sigalet D. L., Martin G. R. Hormonal therapy for short bowel    syndrome. J. Pediatr. Surg. 2000; 35:360-4-   14. Chance W. T., Foley-Nelson T., Thomas I., Balasubramaniam A.    Prevention of parenteral nutrition-induced gut hypoplasia by    coinfusion of glucagon-like peptide-2. 1997; 273:0559-63-   15. Chance W. T., Sheriff S., Foley-Nelson T., Thomas I.,    Balasubramaniam, A. Maintaining gut integrity during parenteral    nutrition of tumor-bearing rats: effects of glucagon-like peptide 2.    Nutrition and Cancer 2000; 37:215-22-   16. Boushey R., Yusta B., Drucker D. J. GLP-2 decreases mortality    and reduces the severity of indomethacin-induced murine enteritis.    Am. J. Physiol. 1999; 277:E937-47-   17. Drucker D. J., Yusta B., Boushey R. P., DeForest L.,    Brubaker P. L. Human [Gly2]GLP-2 reduces the severity of colonic    injury in a murine model of experimental colitis. Am. J. Physiol.    1999; 276:G79-91-   18. Boushey R. P., Yusta B., Drucker D. J. Glucagon-like peptide    (GLP)-2 reduces chemotherapy-associated mortality and enhances    survival in cells expressing a transfected GLP-2 receptor. Cancer    Res. 2001; 61:687-93-   19. Tavakkolizadeh A., Shen R., Abraham P., Kormi N., Seifert P.,    Edelman E. R., et al. Glucagon-like peptide 2: A new treatment for    chemotherapy-induced enteritis. J. Surg. Res. 2000; 91:77-82-   20. Monroe D. G., Gupta A. K., Kooshesh P., Vyas T. B., Rizkalla G.,    Wang H., et al. Prototypic G protein-coupled receptor for the    intestinotrophic factor glucagon-like peptide-2. Proc. Natl. Acad.    Sci. USA 1999; 96:1569-73-   21. Yusta B. Somwar R., Wang F., Munroe D., Grinstein S., Klip A.,    et al. Identification of glucagon-like peptide-2 (GLP-2)-activated    signaling pathways in baby hamster kidney fibroblasts expressing the    rat GLP-2 receptor. J. Biol. Chem. 1999; 274:30459-67-   22. Yusta B., Boushey R. P., Drucker D. J. The glucagon-like    peptide-2 receptor mediates direct inhibition of cellular apoptosis    via a cAMP-dependent protein kinase-independent pathway. J. Biol.    Chem. 2000; 275: 35345-52-   23. Walsh, N. A., Yusta, B., DaCambra, M. P., Anini, Y., Drucker, D.    J., Brubaker, P. L. Glucagon-like peptide-2 receptor activation in    the rat intestinal mucosa. Endocrinology 2003; 144(10):4385-92-   24. Lovshin, J. A., Huang, Q., Seaberg, R., Brubaker, P. L.,    Drucker, D. J. Extrahypothalamic Expression of the Glucagon-like    Peptide-2 (GLP-2) Receptor is Coupled to Reduction of    Glutamate-induced Cell Death in Cultured Hippocampal Cells.    Endocrinology 2004; 145: 3495-506-   25. Burrin D. G., Stoll B., Jiang R., Petersen Y., Elnif J.,    Buddington R. K. et al. GLP-2 stimulates intestinal growth in    premature TPN-fed pigs by suppressing proteolysis and apoptosis.    Am. J. Physiol. 2000; 279:G1249-56-   26. Kouris G. J., Rossi Q. L., Djuricin G., Nathan G. C.    Weinstein R. A., Prinz R. A. The Effect of Glucagon-like Peptide 2    on Intestinal Permeability and Bacterial Translocation in Acute    Necrotizing Pancreatitis. Am. J. Surg. 2001; 181:571-575-   27. Prasad R., Alavi K., Schwartz M Z. Glucagon-like peptide-2    analogue enhances intestinal mucosal mass after ischemia and    reperfusion. J. Pediatr. Surg. 2000; 35:357-9-   28. Prasad R., Alavi K., Schwartz M Z. GLP-2□ accelerates recovery    of mucosal absorptive function after ischemia/reperfusion. J.    Pediatr. Surg. 2001; 36: 570-572-   29. Alavi K., Schwartz Z., Palazzo J. P., Prasad R. Treatment of    inflammatory bowel disease in a rodent model with the intestinal    growth factor glucagon-like peptide-2. J. Ped. Surg. 2000; 35:847-51-   30. Cameron H., Yang P.-C., Perdue M. H. Glucagon-like    Peptide-2-enhanced Barrier Function Reduces Pathophysiology in a    Model of Food Allergy. Am. J. Physiol. 2003; 284: G905-G912-   31. Ramsanahie A. P., Perez A., Duensing A. U., Zinner M. J.,    Ashley S. W., Wang E. E. Glucagon-like peptide 2 enhances intestinal    epithelial restitution. J Surg Res 2002; 107: 44-9

We claim:
 1. A method for determining the ability of a compound topotentially differentially modulate glucagon-like peptide-2 (GLP-2)receptor activity in different sections of the intestine, comprising: a)incubating a first ex vivo intestinal segment with the compound; b)measuring contractility of the first intestinal tissue segment; c)incubating a second ex vivo intestinal tissue segment with the compound;and d) measuring contractility of the second ex vivo intestinal tissuesegment; wherein the first ex vivo intestinal tissue segment is takenfrom a different section of the intestine than the second ex vivosegment, and wherein a difference in contractility of the first ex vivointestinal tissue segment and the second ex vivo intestinal tissuesegment indicates a different responsiveness of the two segments toGLP-2 receptor modulation.
 2. A method according to claim 1, wherein thefirst and second ex vivo intestinal tissue segments are of mammalianorigin.
 3. A method according to claim 2, wherein the first and secondex vivo intestinal tissue segments are of human origin.
 4. A methodaccording to claim 2, wherein the first and second ex vivo intestinaltissue segments are of rat origin.
 5. A method according to claim 1,wherein the first and second ex vivo intestinal tissue segments areselected from the group consisting of duodenum, jejunum, ileum, smallintestine, and colon.
 6. A method for determining the ability of acompound to potentially differentially modulate glucagon-like peptide-2(GLP-2) receptor activity in different sections of the intestine,comprising the steps of: a) obtaining a first ex vivo intestinal tissuesegment, b) obtaining a second ex vivo intestinal tissue segment, c)suspending the first ex vivo intestinal tissue segment and the second exvivo intestinal tissue segment in separate organ baths, d) placing thefirst and second ex vivo intestinal tissue segments under tension, e)incubating each ex vivo intestinal tissue segment with the compound, f)measuring the contractility of the first and second ex vivo intestinaltissue segments, and g) comparing the contractility of the first ex vivointestinal tissue segment with the contractility of the second ex vivointestinal tissue segment; wherein the first ex vivo intestinal tissuesegment is taken from a different section of the intestine than thesecond ex vivo segment, and wherein a difference in contractility of thefirst ex vivo intestinal tissue segment and the second ex vivointestinal tissue segment indicates a different responsiveness of thetwo segments to GLP-2 receptor activation.
 7. A method according toclaim 6, wherein the first and second ex vivo intestinal tissue segmentsare of mammalian origin.
 8. A method according to claim 7, wherein thefirst and second ex vivo intestinal tissue segments are of human origin.9. A method according to claim 7, wherein the first and second ex vivointestinal tissue segments are of rat origin.
 10. A method according toclaim 6, wherein the first and second ex vivo intestinal tissue segmentsare selected from the group consisting of duodenum, jejunum, ileum,small intestine, and colon.
 11. A method for determining the ability ofa compound to differentially antagonize glucagon-like peptide-2 (GLP-2)receptor response to a GLP-2 receptor ligand in different sections ofthe intestine, comprising: a) incubating a first ex vivo intestinaltissue segment with the GLP-2 receptor ligand; b) taking a firstmeasurement of the contractility of the first ex vivo intestinal tissuesegment, c) incubating the first ex vivo intestinal tissue with theGLP-2 receptor ligand and the compound, d) taking a second measurementof contractility of the first intestinal tissue segment, e) incubating asecond ex vivo intestinal tissue segment with the GLP-2 receptor ligand;f) measuring contractility of the second ex vivo intestinal tissuesegment, g) incubating the second ex vivo intestinal tissue with theGLP-2 receptor ligand and the compound, h) taking a second measurementof contractility of the second ex vivo intestinal tissue segment,wherein an increase in the second measurement of contractility relativeto the first measurement of contractility for both the first and secondex vivo intestinal tissue segments is indicative of the compoundpotentially antagonizing the GLP-2 receptor response to the GLP-2receptor ligand in that tissue segment, and wherein a difference inpotential antagonization of of GLP-2 receptor response to the GLP-2receptor ligand in the first ex vivo intestinal tissue and the second exvivo intestinal tissue indicates a differential responsiveness of thetwo regions of the intestine to the ability of the compound toantagonize the GLP-2 receptor response to the GLP-2 receptor ligand. 12.A method according to claim 11, wherein the first and second ex vivointestinal tissue segments are of mammalian origin.
 13. A methodaccording to claim 12, wherein the first and second ex vivo intestinaltissue segments are of human origin.
 14. A method according to claim 12,wherein the first and second ex vivo intestinal tissue segments are ofrat origin.
 15. A method according to claim 11, wherein the first andsecond ex vivo intestinal tissue segments are selected from the groupconsisting of duodenum, jejunum, ileum, small intestine, and colon.